{"doi":"10.1093/nar/gkad044","title":"Nanopore sequencing for N1-methylpseudouridine in RNA reveals sequence-dependent discrimination of the modified nucleotide triphosphate during transcription","abstract":"Direct RNA sequencing with a commercial nanopore platform was used to sequence RNA containing uridine (U), pseudouridine (Ψ) or N1-methylpseudouridine (m1Ψ) in >100 different 5-nucleotide contexts. The base calling data for Ψ or m1Ψ were similar but different from U allowing their detection. Understanding the nanopore signatures for Ψ and m1Ψ enabled a running start T7 RNA polymerase assay to study the selection of UTP versus ΨTP or m1ΨTP competing mixtures in all possible adjacent sequence contexts. A significant sequence context dependency was observed for T7 RNA polymerase with insertion yields for ΨTP versus UTP spanning a range of 20-65%, and m1ΨTP versus UTP producing variable yields that differ by 15-70%. Experiments with SP6 RNA polymerase, as well as chemically-modified triphosphates and DNA templates provide insight to explain the observations. The SP6 polymerase introduced m1ΨTP when competed with UTP with a smaller window of yields (15-30%) across all sequence contexts studied. These results may aid in future efforts that employ RNA polymerases to make therapeutic mRNAs with sub-stoichiometric amounts of m1Ψ.","journal":"Nucleic Acids Research","year":2023,"id":335045,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":20,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9549,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2023-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":765973,"name":"Cynthia J. Burrows","orcid":"0000-0001-7253-8529","position":1,"is_corresponding":false},{"id":765972,"name":"Aaron M. Fleming","orcid":"0000-0002-2000-0310","position":0,"is_corresponding":true}],"reference_count":39,"raw_metadata":null,"created_at":"2026-07-19T01:09:53.742667Z","pmid":"36727474","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}